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Proper physiological function in the pre- and post-natal proximal tubule of the kidney depends upon the acquisition of selective permeability, apical-basolateral epithelial polarity and the expression of key transporters, including those involved in metabolite, toxin and drug handling. Particularly important are the SLC22 family of transporters, including the organic anion transporters Oat1 (originally identified as NKT) and Oat3 as well as the organic cation transporter Oct1. In ex vivo cultures of metanephric mesenchyme (MM; the embryonic progenitor tissue of the nephron) Oat function was evident before completion of nephron segmentation and corresponded with the maturation of tight junctions as measured biochemically by detergent extractability of the tight junction protein, ZO-1. Examination of available time series microarray data sets in the context of development and differentiation of the proximal tubule (derived from both in vivo and in vitro/ex vivo developing nephrons) allowed for correlation of gene expression data to biochemically and functionally defined states of development. This bioinformatic analysis yielded a network of genes with connectivity biased toward Hnf4α (but including Hnf1α, hyaluronic acid-CD44, and notch pathways). Intriguingly, the Oat1 and Oat3 genes were found to have strong temporal co-expression with Hnf4α in the cultured MM supporting the notion of some connection between the transporters and this transcription factor. Taken together with the ChIP-qPCR finding that Hnf4α occupies Oat1, Oat3, and Oct1 proximal promoters in the in vivo differentiating rat kidney, the data suggest a network of genes with Hnf4α at its center plays a role in regulating the terminal differentiation and capacity for drug and toxin handling by the nascent proximal tubule of the kidney.

Epigenetic processes govern prostate cancer (PCa) biology, as evidenced by the dependency of PCa cells on the androgen receptor (AR), a prostate master transcription factor. We generated 268 epigenomic datasets spanning two state transitions—from normal prostate epithelium to localized PCa to metastases—in specimens derived from human tissue. We discovered that reprogrammed AR sites in metastatic PCa are not created de novo; rather, they are prepopulated by the transcription factors FOXA1 and HOXB13 in normal prostate epithelium. Reprogrammed regulatory elements commissioned in metastatic disease hijack latent developmental programs, accessing sites that are implicated in prostate organogenesis. Analysis of reactivated regulatory elements enabled the identification and functional validation of previously unknown metastasis-specific enhancers at HOXB13 , FOXA1 and NKX3-1 . Finally, we observed that prostate lineage-specific regulatory elements were strongly associated with PCa risk heritability and somatic mutation density. Examining prostate biology through an epigenomic lens is fundamental for understanding the mechanisms underlying tumor progression. Analyses of epigenomic datasets spanning transitions from normal prostate epithelium to localized prostate cancer to metastases show that latent developmental programs are reactivated in metastatic disease and that prostate lineage-specific regulatory elements are strongly enriched for prostate cancer risk heritability.

Background: With the rate of kidney disease on the rise, and a serious imbalance between the number of patients requiring a kidney transplant and the number of available donor kidneys, it is becoming increasingly important to develop alternative strategies to restore organ function to diminish the need for human donors. Summary: We review the current progress and future directions of a subset of these strategies which are ultimately aimed towards bioengineering a functional, implantable, kidney-like tissue construct or organoid that might be genetically matched to the patient. Key Messages: By combining the knowledge about normal kidney development with the rapidly growing knowledge in the field of cell differentiation and transdifferentiation, there is hope that partial or complete kidney function can be restored in patients with kidney disease - including genetic disorders, acute kidney injury, or chronic kidney disease - with tissue-engineered construct(s). © 2014 S. Karger AG, Basel [PUBLICATION ABSTRACT]

Proper physiological function in the pre- and post-natal proximal tubule of the kidney depends upon the acquisition of selective permeability, apical-basolateral epithelial polarity and the expression of key transporters, including those involved in metabolite, toxin and drug handling. Particularly important are the SLC22 family of transporters, including the organic anion transporters Oat1 (originally identified as NKT) and Oat3 as well as the organic cation transporter Oct1. In ex vivo cultures of metanephric mesenchyme (MM; the embryonic progenitor tissue of the nephron) Oat function was evident before completion of nephron segmentation and corresponded with the maturation of tight junctions as measured biochemically by detergent extractability of the tight junction protein, ZO-1. Examination of available time series microarray data sets in the context of development and differentiation of the proximal tubule (derived from both in vivo and in vitro/ex vivo developing nephrons) allowed for correlation of gene expression data to biochemically and functionally defined states of development. This bioinformatic analysis yielded a network of genes with connectivity biased toward Hnf4[alpha] (but including Hnf1[alpha], hyaluronic acid-CD44, and notch pathways). Intriguingly, the Oat1 and Oat3 genes were found to have strong temporal co-expression with Hnf4[alpha] in the cultured MM supporting the notion of some connection between the transporters and this transcription factor. Taken together with the ChIP-qPCR finding that Hnf4[alpha] occupies Oat1, Oat3, and Oct1 proximal promoters in the in vivo differentiating rat kidney, the data suggest a network of genes with Hnf4[alpha] at its center plays a role in regulating the terminal differentiation and capacity for drug and toxin handling by the nascent proximal tubule of the kidney.

The proximal tubule (PT) is responsible for more than half of the water reabsorption, recovery of organic solutes, and practically all of the clearance of drugs and metabolites within the kidney. Understanding the transcriptional regulation of PT cells is of major biological and clinical importance, yet much remains to be known. This dissertation attempts to address this problem using multiple molecular, cellular and systems biological approaches. Chapter 2 explores the regulation of drug metabolizing enzyme and transporter (collectively referred to as DMEs) expression in the PT, predominantly focusing on the role of hepatocyte nuclear factor 4a (Hnf4a). Systems analysis revealed hepatocyte nuclear factors Hnf1a and Hnf4a as being potential regulators of DME expression in the PT. Examining genomic localization of Hnf4a and enhancer-associated protein E1A binding protein p300 (p300) by ChIP-sequencing provided additional evidence that Hnf1a and Hnf4a are candidate lineage-determining transcription factors for the proximal tubule cellular identity. A small molecule Hnf4a antagonist was used to show that Hnf4a is required for the expression of multiple DMEs in the kidney proximal tubule in an ex vivo kidney organ culture model. Finally, ectopic expression of Hnf1a and Hnf4a in mouse embryonic fibroblasts (MEFs) was used to show that Hnf1a and Hnf4a can induce the transcription of a number of important DMEs, and established functional organic anion transport capacity – a specific property of the PT. Chapter 3 further explores the capacity of Hnf1a and Hnf4a to establish a PT cell-like identity. We show that while transducing MEFs with Hnf1a and Hnf4a does not completely transdifferentiate them to PT-like cells, they do form tight-junctions in culture and express a broad array of transporters and junctional components, both important characteristics of PT cells. We then show that three hepatocyte lineage-determining factors Gata4, Foxa2 and Foxa3 repress the induction of PT signature genes by Hnf1a and Hnf4a, and in turn upregulate the expression of hepatocyte signature genes, thus identifying a transcriptional switch that appears to help confer tissue-specific roles of Hnf1a and Hnf4a.

Tissue engineering approaches to enhancing or replacing kidney function are of major interest, and in recent years, significant progress has been made at the basic level. A number of methods attempt to recapitulate the aspects of developmental processes that are normally involved in kidney organogenesis. These methods tend to utilize cells and/or progenitor tissues in two- and three-dimensional culture systems, which can be combined to form seemingly functional kidney-like tissue, as judged by immunohistochemistry, gene expression, and transport capacity. When implanted in rodents in vivo, the engineered tissue constructs have been shown to undergo renal developmental processes, recruit vasculature, and in some cases appear to develop characteristics of mature kidney function. In the future, it might be possible to culture an implantable organoid capable of partly or completely replacing kidney function. Here, we discuss some of these methods and their potential applicability in light of recent advances in regenerative medicine.